应用RD-PCR技术制备基因芯片靶基因片段

应用RD－PCR技术分离SH－SY5Y细胞的基因片段。从正常培养的SH－SY5Y细胞中提取总RNA，经oligo(dT）纤维素柱纯化分离出mRNA，然后以oligo(dT18)为锚定引物反转录生成单链cDNA，再以此为模板合成DNA的第二条链；将双链DNA经Sau3AI酶切之后，接上接头，经通用引物和选择性引物进行扩增；然后与载体pMD18-T相连，克隆鉴定、筛选、测序。所分离的cDNA片段经过扩增后用于制备基因芯片的靶基因，杂交检测的结果表明，此种方法所分离的基因片段可以用于基因芯片的靶基因片段，所制备的芯片将为进一步研究神经细胞基因表达提供了条件。

Preparation of target gene fragment of gene chip by method of RD-PCR

A new method was introduced by using the RD-PCR (restriction display PCR) technique in the preparation of target gene fragment of gene chip from SH-SY5Y cell. mRNA was extracted from SH-SY5Y cells. The single strand cDNA was synthesized using an anchored primer oligo(dT18), then the second strand was yielded by random priming. The double strands were cleft with restriction enzyme Sau3AI, then the fragments were ligated with universal adapters. The products of ligation were amplified with the universal primers and selected primers. The products were then ligated into the pMD18-T vector. The recombinant plasmid clones were identified, screened and sequenced. PCR products of interested clones were amplified with primers located in the vector. A Cartesian Microarrayer spotted the amplified cDNA fragments on the amino-modified glass slides. The results of gene chip hybridization shows that the gene fragments prepared by the method of RD-PCR can be used as the target gene fragments for the preparation of gene chip. The gene chip can be used in the study of the gene expression profile of the neurons.